This is a revised application for an R35 grant from the National Cancer Institute entitled, ?Mechanisms of human papillomavirus entry.? HPV is responsible for 5% of human cancer, millions of cases of genital warts, and countless cases of other types of papillomas (most caused by non-vaccine HPV types). Despite the existence of effective vaccines, HPV infection and the cancers it causes will remain a major public health problem for decades because vaccine uptake is poor and vaccination does not clear established infections. There are no specific treatments for HPV or HPV-associated cancers. The DiMaio lab has received more than 35 years of continuous funding from the NCI, during which time it has made seminal contributions to tumor virology. We recently discovered that HPV traffics via the retrograde transport pathway during infection, showed that retromer is required for sorting of the incoming virus particle into this pathway and that HPV is a novel type of retromer cargo, and discovered a cell-penetrating peptide (CPP) that drives the HPV L2 capsid protein into the cytoplasm to engage retromer. This is the first example where either retromer or a CPP has been shown to play a role in virus entry. These results have been paradigm-shifting in the field and have been published since 2013 in Cell, PLoS Pathogens, mBio, PNAS, and Journal of Cell Biology, and they have fundamentally changed our understanding of HPV entry and the role of retromer and CPPs in biology. Here, we will discover how HPV accomplishes these amazing feats. We will determine the requirements for L2 membrane protrusion and establish how sequences flanking the core CPP modulate its activity. We will test whether the abundance of CPP sequences in the extant papillomavirus virome reflects their membrane- penetrating activity, determine whether L2 truly adopts a transmembrane existence, and map L2 segments exposed in the cytoplasm. We have designed an innovative new functional genetics approach and used it to isolate artificial small transmembrane proteins that inhibit HPV entry, and we will use these artificial proteins to identify new HPV entry factors and dissect their role in HPV entry. We will exploit our understanding of HPV entry to design inhibitory peptides that harness the membrane-penetrating activity of CPPs to deliver the retromer binding site into the cytoplasm to compete for binding with incoming HPV, validating an entirely new approach to prevent virus infections. We will develop the first genetic system to select HPV mutants with informative phenotypes, attempt to identify the HPV entry receptor, and extend these studies to additional tumor viruses and HIV. These experiments will elucidate important aspects of the mechanisms of HPV entry, validate new approaches to prevent and treat HPV infection, and revolutionize genetic analysis of HPV. Critically, our studies will provide new insights into fundamental cell biology. If this proposal is funded, we will continue to make novel and important contributions to virology and cancer research for many years.